What are common causes of peak tailing and/or low resolution (broad peaks) in protein-based SEC methods? - WKB230675

ENVIRONMENT

Size exclusion chromatography (SEC)
Peak tailing
ACQUITY
UltraPerformance Liquid Chromatography (UPLC)
Bridged Ethlylene Hybrid (BEH)
MaxPeak Premier
Column

ANSWER

Common causes of peak tailing and/or low resolution include (but are not limited to):

Microbial Contamination.
- This is one of the most common problems in SEC separations.
  - See Guide to Size-Exclusion Chromatography (SEC) of mAb Aggregates, Monomers, and Fragments for a section which discusses the negative impact of microbial contamination on an SEC separation.
Presence of Teflon (PTFE) in the sample flow path.
- Teflon is very hydrophobic, which causes proteins to absorb to it and can result in tailing issues.
- If a Teflon flow cell is used, switch to a stainless steel or titanium flow cell to correct this issue.
System dispersion (bandspread, band broadening) is too high.
- Too much system volume between the injector and the detector leads to wider peaks and, sometimes, tailing.
  - New flow cell which has more volume vs. old flow cell.
  - New tubing, which has more volume vs. old tubing.
- Poor connections in the flow path can lead to peak shape problems, including tailing.
  - Incorrect ferrule depth (too short) can cause voids inside the end fitting.
  - Slipped ferrule (tubing can slip inside the end fitting, causing a void).
Non-specific binding:
- Proteins can adsorb to stainless steel surfaces through ionic attraction to the positive charges on the stainless steel, leading to tailing.
- Proteins can adsorb to the SEC particles by hydrophobic attraction.
  - Make several injections of a biomolecule to saturate stainless steel (non-MaxPeak Premier hardware) and particle active sites to prevent unwanted non-specific adsorption. See the page 3 of the ACQUITY UPLC Protein Protein BEH SEC Columns and Standards Care and Use Manual which describes how to make conditioning injections onto an ACQUITY UPLC Protein BEH SEC Column.
- Waters now offers ACQUITY Premier Protein SEC 250A and XBridge Premier Protein SEC 250A columns, which are designed to prevent non-specific adsorption to stainless steel or the particles.
Accumulation of particulates at the head of the column:
- Microbial growth/contamination:
  - May cause peak tailing but more often causes split peaks, lower resolution, and high-pressure problems.
    - Replace low ionic strength mobile phase (<150 mM) every two to three days.
    - Replace high ionic strength mobile phase (>150mM) every two weeks.
- Excipients (such as polysorbate 80) and other matrix components:
  - May adsorb to the packing which will lower resolution.
Hydrophobic secondary interactions:
- May cause peak tailing.
  - Organic co-solvents are commonly used in SEC mobile phases to mitigate hydrophobic secondary interactions. If an organic modifier is employed, isopropanol (IPA) is the recommended solvent for these columns.
    - Begin with a concentration no greater than 5% of the mobile phase composition and increase, only if ncessary, up to a maximum of 15%.
Column storage in a refrigerator:
- May lead to degradation in column performance.
  - Waters recommends that SEC columns such as the ACQUITY UPLC Protein BEH SEC, XBridge Protein BEH SEC, ACQUITY Premier Protein SEC 250A and XBridge Premier Protein SEC 250A columns not be stored in a refrigerator.
    - Store these columns in 10% acetonitrile/90% 25 mM sodium phosphate pH 7.0 + 100 mM KCl at room temperature instead.
Non-optimal column storage solvent:
- Waters recommends storing these colums in a solution of 10% acetonitrile/90% 25 mM sodium phosphate pH 7.0 + 100 mM KCl at room temperature.
  - If storing in 20% methanol, it is possible that resolution of the High Molecular Weight Species of Low Molecular Weight Species may be adversely affected.
    - See Best Practices for Maintaining Column Performance in Size-Exclusion Chromatography during Long-Term Storage

ADDITIONAL INFORMATION

Also see:

Guidelines for Routine Use and Maintenance of Ultra-Performance Size-Exclusion and Ion-Exchange Chromatography Systems (Technology Brief)
- This document shows examples of:
  - How a flow cell with Teflon leads to peak tailing.
  - How microbial contamination decreases efficiency in SEC.
Guide to Size-Exclusion Chromatography (SEC) of mAb Aggregates, Monomers, and Fragments
- This document discusses:
  - Importance of Developing a Robust LC-based SEC Method:
    - Choosing the Appropriate BEH SEC Column.
    - Method Development.
  - Considerations When SEC Separated Components Tail and/or Resolution Decreases:
    - Connecting an ACQUITY UPLC Protein BEH SEC Column to an LC System.
    - LC System Dispersion Effects on a UPLC SEC Separation.
    - Determining LC System Dispersion Volume.
  - Maximizing Protein BEH SEC Column Life:
    - Mobile Phase Preparation and Use.
    - Avoiding Microbial Contamination of the Solvent Delivery System.
    - Cleaning ACQUITY UPLC Systems.
    - Minimizing Sample Particulate Contamination of Protein BEH SEC Columns.
    - Preventing Sample Formulation Constituent Contamination of Protein BEH SEC Columns.
    - Column Storage.
Evaluating the Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Proteins
- This document discusses:
  - Understanding the measurement of LC System Dispersion.
  - An Educational and Systematic demonstration of the impact of LC system dispersion on SEC-based protein separations.
  - Guidance for selecting the optimal SEC column configuration based on:
    1. The LC systems to be used.
    2. The analytical method requirements including:
      - Resolution
      - Sensitivity
      - Reproducibiliy
      - Transferability
Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Monoclonal IgG Antibody Aggregates and Fragments: Selecting the Optimal Column Configuration for Your Method
- This document discusses:
  - An Educational and Systematic demonstration of the impact of LC system dispersion on SEC-based mAb separations.
  - Guidance for selecting the optimal SEC column configuration based on:
    1. The LC systems to be used.
    2. The analytical method requirements including:
      - Resolution
      - Sensitivity
      - Reproducibiliy
      - Transferability
  - A comparison of the SEC separation performance of the ACQUITY UPLC H-Class Bio (UPLC) and ACQUITY Arc Bio (UHPLC) Systems.

Videos:

Do you have to change aqueous mobile phase every day? | Trust your Science 7

Highlights how microbial growth can contaminate system in ~2 days and disrupt chromatography.)

Attack of the column killing aqueous mobile phase bacteria! | Trust Your Science 18

Highlights how a green laser can help you “see” bacterial growth in mobile phase. Shows impact of microbial contamination on SEC column.

MaxPeak Premier Columns:

BioResolve SEC mAb Columns:

ACQUITY UPLC BEH SEC and XBridge BEH SEC Columns:

id230675, eluent, monoclonal antibody